Inadequate antigen presentation and activation of innate and adaptive immunity in humans result in the failure of the human immune system to control and clear many pathogenic infections and malignant cell growth. Successful therapeutic vaccines and immunotherapies for chronic infection and cancer rely on the development of new approaches for efficient means to induce a vigorous immune response which is capable of controlling and clearing offensive antigens associated with their pathologies.
The ability of T cells to recognize an antigen is dependent on the association of the antigen with either major histocompatibility complex (MHC) I or MHC II proteins. For example, cytotoxic T cells respond to an antigen that is presented in association with MHC-I proteins. Thus, a cytotoxic T cell that should kill virus-infected cell will not kill that cell if the cell does not also express the appropriate MHC-I protein. Helper T cells recognize MHC-II proteins. Helper T cell activity depends, in general, on both the recognition of the antigen on antigen presenting cells and the presence on these cells of “self” MHC-II proteins. The requirement for recognition of an antigen in association with a self-MHC protein is called MHC restriction. MHC-I proteins are found on the surface of virtually all nucleated cells. MHC-II proteins are found on the surface of certain cells including macrophages, B cells, and dendritic cells of the spleen and Langerhans cells of the skin.
A crucial step in mounting an immune response in mammals is the activation of CD4+ helper T-cells that recognize MHC-II restricted exogenous antigens. These antigens are captured and processed in the cellular endosomal pathway in antigen presenting cells, such as dendritic cells (DCs). In the endosome and lysosome, the antigen is processed into small antigenic peptides that are complexed onto the MHC-II in the Golgi compartment to form an antigen-MHC-II complex. This complex is expressed on the cell surface, which expression induces the activation of CD4+ T cells.
Other crucial events in the induction of an effective immune response in a mammal involve the activation of CD8+ T-cells and B cells. CD8+ cells are activated when the desired protein is routed through the cell in such a manner so as to be presented on the cell surface as a processed protein, which is complexed with MHC-I antigens. B cells can interact with the antigen via their surface immunoglobulins (IgM and IgD) without the need for MHC proteins. However, the activation of the CD4+ T-cells stimulates all arms of the immune system. Upon activation, CD4+ T-cells (helper T cells) produce interleukins. These interleukins help activate the other arms of the immune system. For example, helper T cells produce interleukin-4 (IL-4) and interleukin-5 (IL-5), which help B cells produce antibodies; interleukin-2 (IL-2), which activates CD4+ and CD8+ T-cells; and gamma interferon, which activates macrophages. Since helper T-cells that recognize MHC-II restricted antigens play a central role in the activation and clonal expansion of cytotoxic T-cells, macrophages, natural killer cells and B cells, the initial event of activating the helper T cells in response to an antigen is crucial for the induction of an effective immune response directed against that antigen. Attempts to stimulate helper T-cell activation using a sequence derived from the lysosomal transmembrane proteins have been reported. However, these attempts did not result in the induction of effective immune responses with respect to CD8+ T-cells and B cells in the mammals being tested.
In addition to the critical roles that T cells play in the immune response, DCs are equally important. DCs are professional antigen-presenting cells having a key regulatory role in the maintenance of tolerance to self-antigens and in the activation of innate and adaptive immunity (Banchereau et al., 1998, Nature 392:245-52; Steinman et al., 2003, Annu. Rev. Immunol. 21:685-711). When DCs encounter pro-inflammatory stimuli such as microbial products, the maturation process of the cell is initiated by up-regulating cell surface expressed antigenic peptide-loaded MHC molecules and co-stimulatory molecules. Following maturation and homing to local lymph nodes, DCs establish contact with T cells by forming an immunological synapse, where the T cell receptor (TCR) and co-stimulatory molecules congregate in a central area surrounded by adhesion molecules (Dustin et al., 2000, Nat. Immunol. 1:23-9). Once activated, CD8+ T cells can autonomously proliferate for several generations and acquire cytotoxic function without further antigenic stimulation (Kaech et al., 2001, Nat. Immunol. 2:415-22; van Stipdonk et al., 2001, Nat. Immunol. 2:423-9). It has therefore been proposed that the level and duration of peptide-MHC complexes (signal 1) and co-stimulatory molecules (signal 2) provided by DCs are essential for determining the magnitude and fate of an antigen-specific T cell response (Lanzavecchia et al., 2001, Nat. Immunol. 2:487-92; Gett et al., 2003, Nat. Immunol. 4:355-60).
Antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages, play important roles in the activation of innate and adaptive immunity as well as in the maintenance of immunological tolerance. The mechanisms by which APCs sense microbes and initiate immune responses has been well studied during the last several years. APCs use pattern-recognition receptors such as toll-like receptors (TLRs) to recognize conserved microbial structures such as lipopolysaccharide (LPS), unmethylated bacterial DNA (CpG), and RNA. TLRs belong to the TIR (Toll/interleukin-1 (IL-1) receptor) superfamily, which is divided into two main subgroups: the IL-1 receptors and the TLRs. TLRs consist of 11 members (TLR1-TLR11). All members of this superfamily signal in a similar manner due to the presence of a conserved, cytosolic TIR domain, which activates common signaling pathways, especially those leading to the activation of the transcription factor nuclear factor-κB (NF-κB) and stress-activated protein kinases. NF-κB activation catalyzes immune responses by secreting proinflammatory cytokines such as tumor necrosis factor (TNF), IFN, interleukin 1 (IL-1), IL-6, and IL-12 and by expressing costimulatory molecules such as CD80, CD86, and CD40. The members of TNF family, such as TNFα and CD40L, intereact with their receptors or ligands and also activate NF-κB.
Major efforts to develop tumor vaccines have attempted to promote DC maturation and costimulation as a means of enhancing antitumor immunity. However, the induction of immunity against self tumor-associated antigens (TAAs) is restricted by intrinsic inhibitory mechanisms, many of which remain to be defined. A known inhibitory mechanism is employed by cytotoxic T-lymphocyte antigen 4 (CTLA4) and related molecules on T-cells to control the magnitude of effector T-cell activation via cell-cell contact with B7 family molecules on DCs or other cells. DC maturation serves as the critical switch from the maintenance of self-tolerance to the induction of immunity. However, it remains unclear whether mature antigen-presenting DCs possess a negative immune regulatory mechanism that would allow them to control the magnitude and duration of adaptive immunity beyond the point of maturation.
Much attention has focused on pro-inflammatory signaling but little is known about the mechanisms that suppress and resolve inflammation. The magnitude and duration of TLR-initiated immune responses is dictated by the strength and duration of proinflammatory signaling and by the regulation of signal transduction pathways. Since TLR-induced activation of the transcription factor NF-κB is essential for the transcription of a large number of proinflammatory genes, multiple mechanisms are utilized to negatively regulate TLR signaling at multiple levels for the protection of hosts from excessive immune responses such as septic shock and for maintaining immune homeostasis in situations of chronic microbial exposure such as the intestinal microenvironment. Negative immune regulators of the TLR signal pathway include IRAK-M, MyD88s, PI3K, TOLLIP, A20, TRIAD3A, NOD2, soluble TLR2/4, and membrane-bound molecules that contain a TIR domain such as SIGIRR and ST2 (Nat Rev. Immunol. 5:446, 2005).
Cytokines are critically involved in the regulation of multiple immune cell functions (Curtsinger et al., 2003, J. Exp. Med. 197:1141-51; Valenzuela et al., 2002, J. Immunol. 169:6842-9). DCs use toll-like receptors (TLRs), which recognize conserved microbial structures such as lipopolysaccharide (LPS), to promote DC maturation by activating the nuclear factor-κB (NF-κB) signaling pathway (Akira et al., 2004, Nat. Rev. Immunol. 4:499-511). NF-κB family members then mediate the expression of pro-inflammatory cytokines, such as IL-12, resulting in the induction of innate and adaptive immunity (Akira et al., 2004, Nat. Rev. Immunol. 4:499-511; Beutler et al., 2003, Nat. Rev. Immunol. 3:169-76; Janeway et al., 2002, Annu. Rev. Immunol. 20:197-216). Following DC maturation, cytokine production and intracellular signaling pathways are thought to be tightly regulated to promote beneficial immune responses against foreign antigens while limiting excessive autoimmune activation. However, the importance of specific feedback inhibition mechanisms for these pathways and the resulting control of self-antigen specific immune responses remain poorly defined.
SOCS1 is an inducible negative feedback regulator of signaling by various cytokines including interferon (IFN)-γ, interleukin (IL)-2, IL-6, IL-7, IL-12 and IL-15 (Kubo et al., 2003, Nat. Immunol. 4:1169-76; Alexander et al., 2004, Annu. Rev. Immunol. 22:503-29). SOCS1 suppresses multiple signal transducer and activator of transcription (STAT) signaling pathways by binding to the activation loop of the upstream Janus kinases (JAKs) as a pseudosubstrate inhibitor and/or targeting JAK for proteasomal degradation (Kubo et al., 2003, Nat. Immunol. 4:1169-76; Alexander et al., 2004, Annu. Rev. Immunol. 22:503-29). SOCS1 also blocks NF-κB signaling by targeting p65 protein for ubiquitin-mediated proteolysis through its SOCS Box region (Ryo et al., 2003, Mol. Cell. 12:1413-26). SOCS1-deficient (−/−) mice die as neonates with severe systemic inflammation and aberrant activation of T and NKT cells, mainly as a result of unbridled cytokine signaling (Marine et al., 1999, Cell 98:609-16; Alexander et al., 1999, Cell 98:597-608; Naka et al., 2001, Immunity 14:535-45). Although little is known about SOCS1 functions in DCs, recent studies suggest a role for SOCS1 in controlling signaling in antigen presenting cells (APCs) (Kubo et al., 2003, Nat. Immunol. 4:1169-76; Hanada et al., 2003, Immunity 19:437-50). SOCS1 expression in macrophages is induced by LPS or CpG-DNA stimulation and SOCS1−/− mice are more sensitive to LPS-induced shock than are their wild-type littermates (Crespo et al., 2000, Biochem. J. 349:99-104; Dalpke et al., 2001, J. Immunol. 166:7082-9; Nakagawa et al., 2002, Immunity 17:677-87; Kinjyo et al., 2002, Immunity 17:583-91). Moreover, SOCS1−/− DCs from mice in which SOCS1 expression has been restored in T and B cells on a SOCS1−/− background are hyper-responsive to IFNγ and LPS, trigger allogeneic T cell expansion and induce aberrant expansion of B cells and autoreactive antibody production (Hanada et al., 2003, Immunity 19:437-50).
Although little is known about SOCS1 functions in DCs, a recent study has demonstrated a role of SOCS1 in regulating cytokine signaling transduction pathways. For example, it has been demonstrated that SOCS1−/− DCs exhibited a more mature phenotype and were observed to be hyperresponsive to lipopolysaccharide (LPS), which interacts with Toll-like receptor (TLR) 4 for signaling. Also observed was that SOCS1−/− DCs induced autoreactive antibody production. These observations hinted at a possible role for SOCS1 in the negative regulation of DCs possibly by controlling the JAK/STAT pathway and the TLR/NF-κB pathway.
There have been many attempts made to use DCs in immunotherapy to stimulate the immune response in a mammal. In these efforts, DCs were manipulated by loading them with antigen and causing them to mature in an ex vivo context so that they stimulate anti-tumor immunity in a cancer patient. With respect to the use of immunotherapy to combat human immunodeficiency virus (HIV), no effective human immunodeficiency virus (HIV) vaccine has yet emerged. Thus, there is a long felt need in the art for efficient and directed means of eliciting an immune response for the treatment of diseases in mammals. The present invention satisfies this need.